As we have reported previously, both DNA and tRNA become hypomethylated in livers of rats fed a cancer promoting, methyl-deficient diet (MDD) for as short a period as one week. Within the same period, activities of tRNA and DNA methyltransferases (MTases) increase and levels of mRNAs for several genes believed to have roles in growth regulation are altered. These diet-induced changes in nucleic acid methylation and gene expression increased in extent when MDD was fed continuously for four weeks. We also observed hypomethylation of specific CCGG sites within several genes for which mRNA levels were increased. These included c-myc, c-fos and c-Ha-ras. To investigate the reversibility of such diet-induced alterations in methylation and gene expression, animals were fed MDD for four weeks, after which a diet supplemented with adequate sources of methyl groups (CSD) was fed for 1-3 weeks. One to two weeks after the restoration of an adequate diet, the overall extent of methylation of tRNA and DNA from livers of these rats did not differ from that of tRNA and DNA from livers of age matched animals continually maintained on CSD. At the same time, activities of MTases in the liver dropped to normal values. Levels of mRNAs for all genes studied returned to control levels within three weeks after ending MDD feeding, although at different rates. In contrast, MDD-induced hypomethylation of some HpaII sites in c-myc, c-fos and c-Ha-ras genes persisted after 3 weeks refeeding of an adequate diet. These results, which demonstrate that most of the effects of MDD on the parameters we have studied occur rapidly and are essentially reversible, are consistent with the role of MDDs as promoters of hepatocarcinogenesis. However, the finding that unmethylated sites persist in genes that play a role in growth regulation suggests a mechanism by which intermittent or long term exposure to MDDs could result in heritable phenotypic changes in some hepatocytes that lead to hyperplasia and tumorigenesis.
Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In ver-tebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or blockde novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.In vertebrate cells, -3% of cytosine (C) residues in DNA have a methyl group on carbon 5, and 5-methylcytosine (5MeC) is the only naturally occurring modified base detected in DNA (1). Enzymatic methylation of C residues in DNA occurs postreplicatively and primarily involves C residues in CpG dinucleotides, although methylation has been observed at C residues 5' of other nucleotides (2). The extent and pattern of methylation of genomic DNA is species-and tissue-specific (3), which implies that the pattern of methylation is faithfully inherited in all cells of common lineage within a tissue. Analysis of methylation patterns of specific genes during development suggests that patterns established in sperm and oocytes are lost during early development, that regions other than CpG islands become almost fully methylated, and that loss of methylation occurs at specific sites in tissues where a gene is expressed (4-6).Although not all genes are regulated by methylation, hypomethylation at specific sites or in specific regions in a number of genes is correlated with active transcription (7)(8)(9). DNA methylation in vitro can prevent efficient transcription of genes in cell-free systems or transient expression of transfected genes; methylation of C residues in some specific cis-regulatory regions can also block or enhance binding of transcription factors or repressors (7-11). DNA methylation is involved in inactivation of one of the two X chromosomes in female mammalian somatic cells (12), and allele-specific methylation has been proposed as a factor in genomic imprinting (13). The most direct evidence for the importance of DNA methylation in de...
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